Foot-and-mouth disease is a highly contagious disease of great economic importance, afflicting primarily cloven-hoofed animals. The mortality directly attributable to foot-and-mouth disease is comparatively low, generally, but in young animals the mortality can be quite high. Of greater economic importance, the disease is so debilitating that infected animals cannot be raised and fed economically . The only recognized effective procedure for eliminating the infection once it has been discovered is to destroy all infected animals, disinfect all premises which have been occupied by the animals, and decompose the carcasses in quicklime. Since the infection spreads extremely rapidly, the economic foundation of entire communities or regions can be destroyed by one major outbreak of foot-and-mouth disease.
Vaccines have been produced which immunize against foot-and-mouth disease, primarily, by inactivation or attenuation of the virus. Such vaccines have been found to be effective in some measure, but outbreaks of foot-and-mouth disease have been linked to vaccines in which the virus was incompletely inactivated or insufficiently attenuated as well. Infections have also been traced to the escape of virus from facilities devoted to research on foot-and-mouth disease or to production of foot-and-mouth disease vaccines.
Foot-and-mouth disease (FMD) is cuased by a Picornavirus of the genus aphthovirus. There are several viral serotypes of foot-and-mouth disease virus (FMDV), the most common of which are identified by the serotype designation A, O and C, and less common identified as SAT-1, SAT-2, SAT-3 and ASIA-1. Among these serotypes, several subtypes and subtype strains have also been identified. The following are among the identified subtypes and subtype strains: FMDV A, subtype 10, strain 61 and subtype 12, strains 119, USA and Pirbright; FMDV 0, subtype 1, strain Kaufbeuren; and FMDV C, subtype 3, strain Indaial.
FMDV has been described in some detail; see, for example, H. L. Backrach, in Beltsville Symposium on Agricultural Research, J. A. Romberger, Ed., Allanheld, Montclair, N.J. 1977), pp. 3-32; Annual Reviews of Microbiology, 22, 201 (1968). The molecular biology of these viruses have been described, R. R. Rueckert, in Molecular Biology of Picornavizuses, R. Perex-Bercoff, Ed. Plenum, New York, (1979), p. 113. The virus has a molecular size of about 7.times.10.sup.6 daltons and contains a plus-stranded RNA genome of approximately 8,000 nucleotides. Picornavirus proteins have been synthesized in infected cells as a precursor of a protein that is subsequently processed by cellular and virus-coded proteases into four major capsid proteins (VP.sub.1, VP.sub.2, VP.sub.3, and VP.sub.4) and numerous non-capsid proteins.
The whole VP.sub.3 protein when used to inoculate swine elicited a neutralizing anti-body response and protected both swine and cattle from infection. [J. Laporte, et al., C.R. Acad. Sci., 276: 3399 (1973); H. L. Backrach, et al., J. Immunol., 115: 1636 (1975). See also U.S. Pat. No. 4,140,763.] Based upon this information, Dennis G. Kleid, et al. Science, 214: 1125-1129 (Dec. 4, 1981), were able to produce a cloned viral protein vaccine for foot-and-mouth disease which gave antibody responses in cattle and swine.
It is noted that the literature in this field utilizes the same names to refer to different capsid proteins. Thus, the above-mentioned workers in the United States typically refer to the capsid protein referred to herein and in Europe as VP.sub.1, as the VP.sub.3 capsid. There is agreement, however, that the capsid protein referred to herein as VP.sub.1, and referred to by others as VP.sub.3, is the immunologically active capsid protein.
Recombinant DNA molecules and processes for producing peptides with the specificity of foot-and-mouth disease viral antigens are described in United Kingdom Patent Application GB No. 2,079, 288A, Jan. 20, 1982. See also Boothroyd et al, Nature, 290: 800-802 (1981); Kleid et al., Science, 214: 1125-1129 (1981); and EPO Publication Number 0 068 693 2A corresponding to application number 82303040.8 filed 11.06.82.
K. Strohmaier et al., Proc. 5th Int. Congress Virology, Strasbourg, 1981, poster session, have digested the VP.sub.1 protein (denominated VP.sub.Thr therein) with enzymes as well as cyanogen bromide, and raised neutralizing antibodies using the peptide fragments of those digests. Those authors suggested that the amino acid residue sequences at postions 146 through 155 and 200 through 213 from the protein amino-terminus induced production of immunologically important antibodies. Those authors also suggested that amino acid residue sequences at positions 141 through 145 and 155 through 161 were among the regions of inactive, non-inducing peptides. This VP.sub.1 sequence corresponds to the VP.sub.3 sequence described earlier in the United States; see explanation by Meloen, A. H., J. Gen. Virol, 45:761-763 (1979).
A full paper by Strohmaier et al., J. Gen. Virol., 59:295-306 (1982), detailed the work reported at the above poster session, and provides a correlation for the various capsid protein nomenclatures utilized by workers in this field. This paper reiterated the findings reported at the poster session that two cyanogen bromide cleavage products termed CB.sub.1 and CB.sub.2 and an enzyme cleavage product termed A.sub.2 of VP.sub.1 which correspond to amino acid residue positions 55-180, 181-213, and 146-213, respectively, from the amino-terminus, produced neutralizing antibodies. This paper also reiterated that regions of overlap with other cleavage products, including regions 141-145 and, 155-161, had no apparent effect. Those authors stated, at page 303, that they though it "likely that only two small regions are essential for the immunizing potency of the protein . . . "
The poliomyelitis (hereinafter polio) and Hepatitis A viruses are also members; i.e. genera, of the Picornavirus family. Successful vaccines against types 1, 2 and 3 polio viruses have been used since the 1950's, while no successful vaccine against Hepatitis A is known.
One of the distinguishing features of the Picornaviruses is that they contain four capsid proteins. The capsid protein denominated VP.sub.1 of polio type 1 has been found to contain an antigenic determinant region capable of inducing production of antibodies that neutralize the virus, although heretofore the specific amino acid determinant regions of the VP.sub.1 capsid have not been found. A specific capsid of the Hepatitis A virus has not yet been identified as being responsible for inducing production of neutralizing antibodies.
The antipolio vaccines typically utilize inactivated types 1, 2 and 3 viruses. In some instances, all of the allegedly killed viruses have not been killed, or the virus particles have not been sufficiently attenuated, so that about one out of one million innoculations causes an inoculated person to contract clinical disease.
It would therefore be beneficial if an antipolio vaccine could be prepared that is free from any possibility of containing a live or even attenuated virus. It would also be beneficial if a useful antipolio vaccine could be prepared that is free from celluar debris, bacterial endotoxins and growth medium by-products as are frequently present in vaccine preparations obtained from recombinant DNA technology, as is discussed hereinafter. It would be still more beneficial if vaccines and diagnostic products could be found that were safe and highly effective.
In the past antigens have been obtained in several fashions, including derivation from natural materials, coupling of a hapten to a carrier, and by recombinant DNA technology Sela, et al., Proc. Nat. Acad. Sci., U.S.A., 68:1450-1455 (July, 1971); Science, 166:1365-1374 (December 1960); Adv. Immun., 5:29-129 (1966) have also described certain synthetic antigens.
Antigens derived from natural materials are the countless number of known antigens which occur naturally, such as blood group antigens, HLA antigens differentiation antigens, viral and bacterial antigens, and the like. Considerable effort has been expended over the last century in identifying and studying these antigens.
Certain "synthetic" antigens have been prepared by coupling small molecules to carriers such as, for example, bovine serum albumin, thus producing antigens which will cause production of antibody to the coupled small molecule. The carrier molecule is necessary because the small molecule itself would not be "recognized" by the immune system of the animal into which it was injected. This technique has also been employed in isolated instances to prepare antigens by coupling peptide fragments of known proteins to carriers, as described in the above-referenced Sela et al. articles.
While this hapten-carrier technique has served the research community well in its investigations of the nature of the immune response, it has not been of significant use to produce antigens which would play a role in diagnostic or therapeutic modalities. The reasons for this deficiency are several.
First, to choose and construct a useful antigenic determinant from a pathogen by this technique, one must determine the entire protein sequence of the pathogen to have a reasonable chance of success. Because of the difficulty of this task it has rarely, if ever, been done
Classically, vaccines are manufactured by introducing killed or attenuated organisms into the host along with suitable adjuvants to initiate the normal immune response to the organisms while, desirably, avoiding the pathogenic effects of the organism in the host. The approach suffers from the well known limitations in that it is rarely possible to avoid the pathogenic response because of the complexity of the vaccine which includes not only the antigenic determinant of interest but many related and unrelated deleterious materials, any number of which may, in some or all individuals, induce an undesirable reaction in the host.
For example, vaccines produced in the classical way may include competing antigens which are detrimental to the desired immune response, antigens which include unrelated immune responses, nucleic acids from the organism or culture, endotoxins and constituents of unknown composition and source. These vaccines, generated from complex materials, inherently have a relatively high probability of inducing competing responses even from the antigen of interest. In addition, such known vaccines against FMDV must be kept refrigerated prior to use, and refrigeration in remote areas where the vaccines are used is often difficult to obtain.
Recombinant DNA technology has opened new approaches to vaccine technology which does have the advantage that the manufacture begins with a monospecific gene; however, much of this advantage is lost in actual production of antigen in Escherichia coli, or other micro organisms. In this procedure, the gene material is introduced into a plasmid which is then introduced into E. coli which produces the desired protein, along with other products of the metabolism, all in mixture with the nutrient. This approach is complicated by the uncertainty whether the desired protein will be expressed in the transformed E. coli.
Further, even though the desired protein may be produced, there is uncertainty as to whether or not it can be harvested, or whether it will be destroyed, in the process of E. coli growth. It is well known, for example, that foreign or altered proteins are digested by E. coli. Even if the protein is present in sufficient quantities to be of interest, it must still be separated from all of the other products of the E. coli metabolism, including such deleterious substances as undesired proteins, endotoxins, nucleic acids, genes and unknown or unpredictable substances.
Finally, even if it were possible, or became possible through advanced, though necessarily very expensive, techniques, to separate the desired protein from all other products of the E. coli metabolism, the vaccine still comprises an entire protein which may include undesirable antigenic determinants, some of which are known to initiate very serious, adverse responses. Indeed, it is known that certain proteins which could otherwise be considered as vaccines include an antigenic determinant which induces such serious cross reference or side reactions as to prevent the use of the material as a vaccine.
It is also possible, using hybridoma technology, to produce antibodies to viral gene products. Basically, hybridoma technology allows one to begin with a complex mixture of antigens and to produce monospecific antibodies later in the process. In contrast, the present invention is the reverse process, in that it starts with the ultimate in high purity antigenic determinant and thus avoids the necessity for purification of the desired antigenic product.
Hybridoma antibodies are known to be of low avidity and low binding constant, and therefore, have limited value.
Ultimately, in hybridoma technology, one must rely on the production of the antibody by cells which are malignant, with all of the attendant concerns regarding separation techniques, purity and safety.
Hybridoma production relies upon tissue culture or introduction into mice, with the obvious result that production is costly; there is also inherent variability from lot to lot.
In addition, it is difficult to make a hybrid to molecules which comprise only a small percentage of the complex mixture one must start with.
Previous studies by Arnon et al., Proc. Nat. Acad. Sci. U.S.A. 68:1450 (1971), Atassi, Immunochemistry 12:423 (1975) and Vyas et al., Science 178:1300 (1972) have been interpreted by those authors to indicate that short linear amino acid sequences are, in general, unlikely to elicit antibodies reactive with the native protein structure. It was though that for most regions of most molecules, antigenic determinants resulted from amino acid residues well separated in the linear sequence but conformation of the peptides used to elicit antibodies was thought to be critical in most cases, even for those antigens involving amino acides close together in a sequence. Lerner, et al., Cell 23:109-110, (1981); Nature 287:801-805 (1980), discovered that antibodies to linear peptides react with native molecules. Elaborate biosyntheses thus become unnecessary, uneconomical and obsolete.
Nothwithstanding the availability of inactivated or attenuated virus vaccines against foot-and-mouth disease, there has remained a great economic and practical demand for, and great theoretical interest, in the development of a vaccine against foot-and-mouth disease which would be free of the risks which have heretofore attended the manufacture and handling of the FMDV which causes the disease. The availability of cloned viral proteins may well be a very significant step forward from the older and very risky approaches.
However, the cloned viral protein vaccine approach also carried with it a number of inherent disadvantages, limitations and risks. Variations in the biosynthesis system itself may cause variation in expression of proteins, thus affecting purity, yields, potency, etc. of antigens. In addition, the presence of other proteins, and difficult and inefficient separations, suggest the likelihood that vaccines produced through the cloned viral protein route will not be monospecific. Thus, purity, potency, and safety are major concerns with products derived from this technology.
Nothwithstanding that the general concept of preparing synthetic antigens, starting either from a known peptide sequence or from a genome have been described, and notwithstanding that the synthesis of peptides of suitable length for use in antigenic materials is now quite well known, there remains a very large area of antigen-antibody technology which continues to defy predictabilty. While there are some guidelines and some suggestions as to possible antigenic sequences, the field remains largely a matter of speculation, and of trial and error. Even with the recognition that a long sequence may contain antigenicaly active constituents, there remains a great deal of uncertainty and speculation as to whether all or only part of the sequence is required for antigenicity, and whether or not a smaller portion of the sequence would be of greater or lesser antigenicity.